Development of the solid-phase extraction protocol

The previous method of purification for glucuronides from the glucuronylsynthase reaction employed normal- or reverse-phase chromatography with a mobile phase of either 7:2:1 ethyl acetate:methanol:water or 25% aqueous acetonitrile with 0.1% formic acid. As might be expected on analytical scales these chromatographic methods were time-consuming and resulted in the loss of material or contamination with silica gel to give inaccurate yields.

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Therefore a different purification method was sought to simplify the approach on analytical scales and solid-phase extraction was selected as the most viable method to this end.

2.2.1.1 Principles of solid-phase extraction

It is useful at this point to discuss the principles of solid-phase extraction. Solid-phase extraction (hereafter abbreviated as SPE) is an analytical technique commonly used in anti-doping laboratories for quick and efficient extraction of compounds of interest from a matrix such as urine. It is commonly used alongside the other well-known analytical techniques (especially in anti-doping laboratories) such as gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS)75_.

What differentiates SPE from standard chromatography methods is that the separation of compounds from a mixture by SPE is dependent upon the differing chemical properties of the compound and their resulting absorption onto a solid support (sorbent), whereas by flash column chromatography the separation depends upon the retention of the compounds on silica. Typically, SPE is performed in cartridge form as opposed to the use of a glass column and can be used as an array of cartridges when high-throughput purification is required.

The sorbent for a cartridge has various functional groups attached, with some having high affinity for anions, others for cations, and still others for lipophilic/hydrophilic interactions, depending on the type of cartridge used. Through the use of carefully chosen mobile phases during elution it is theoretically possible to separate all or most of the components selectively with very little if any contamination.

As an example of how SPE works, figure 8 illustrates the extraction of DHEA 3-glucuronide 21 directly from a reaction mixture by the use of an Oasis WAX (weak anion exchange) cartridge, referred to as a mixed mode polymeric, weak anion exchange system. The reaction mixture contains the parent steroid 19, α-D-glucuronyl fluoride 29 (with possible hydrolysis product 6),

the glucuronide product 21, and the glucuronylsynthase enzyme.

There are three key components present on the sorbent of the cartridge. The first component is the polymeric hydrocarbon functionality (green), which provides the lipophilic interaction with the similarly lipophilic backbone of the steroid. The second component is the hydrophilic, neutral functionality (red), such as the γ-lactam, which provides the polar interactions with the hydroxyl groups on the sugar.

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Figure 8: the chemistry of an Oasis WAX SPE cartridge, illustrating the purification method for a

crude glucuronylsynthase reaction of DHEA 19, with the corresponding functionalities on the sorbent, colour-coded for clarity

The third and perhaps the most important component is the ionic functionality (blue) of piperazine, or more specifically the piperazinium cation, which is generated by the initial pre- conditioning of methanol followed by water to protonate piperazine, which is sufficiently basic at about pH 6-7. This provides the necessary ionic interaction with the carboxylate moiety of the sugar, also charged at pH 6-7, allowing it to trap the sugar and the glucuronide.

The first mobile phase (in this example; other mobile phases may be used depending on requirements) is 2% v/v aqueous formic acid, which serves to elute the sugar(s) and any salts selectively by protonating the carboxylate group. This may also protonate the same group on the glucuronide 21, but since this leads to an effectively neutral organic compound the glucuronide (and the parent steroid 19) does not partition favourably into the mobile phase, while the sugar is readily eluted.

Once the aqueous formic acid elution is completed, the cartridge is then washed thoroughly with water, which returns the pH of the mobile phase to about 6-7, thereby re-ionising the carboxylate on the glucuronide 21.

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Elution with methanol is then performed. The parent steroid 19 is readily soluble in methanol, as is the glucuronide 21. However, as the glucuronide 21 exhibits a charged interaction with the stationary phase it does not partition into the mobile phase, while the parent steroid 19 is readily eluted.

All that remains to be done is to elute the glucuronide 21 from the sorbent. This is achieved by using 5% v/v ammonium hydroxide in methanol as the mobile phase. Here, ammonium hydroxide deprotonates the piperazinium cations, removing the charged interaction and allowing the glucuronide to partition into methanol and elute from the SPE cartridge in the form of an ammonium salt.

There are many different cartridge types that may be used depending on the components present in the mixture of interest, and manufacturers may publish some recommended methodologies for their cartridges.

2.2.2.2 SPE cartridge evaluation

For evaluation of solid phase extraction as a rapid purification technique for the glucuronylsynthase reaction, two cartridges for SPE that were on hand in our laboratory were trialled, namely the Oasis WAX cartridge (as previously described) and the Bond Elut Certify II cartridge (manufactured by Varian), both of which were mixed-mode reverse-phase anion exchange cartridges. A number of protocols were also examined for use with these cartridges. These were the Oasis WAX and the Australian Racing Forensic laboratories (AFRL) protocols for the WAX cartridge, and the Oasis MAX, ARFL, and manufacturer’s protocols for the Bond Elut Certify II cartridge (figure 9).

A concentration of 0.69 mM stock solution in buffer and 10% (v/v) tert-butanol was prepared for both DHEA 19 and DHEA 3-glucuronide 21 in a 1:1 mixture used to simulate an enzyme reaction where ~50% conversion had occurred.

The first cartridge, the Oasis WAX cartridge, uses mixed-mode reverse-phase sorbent containing polar and weak anion exchange functionalities (as seen previously in figure 8). Using the WAX protocol, we initially observed that DHEA 3-glucuronide 21 was being eluted in the flow-through (solvent from loading the enzyme reaction on the cartridge). In fact, we could only load about 1% of the resin mass – for example, the WAX cartridge has a stated resin mass of 60 mg – and so we were only able to separate ~0.5 mg at a time.

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However, once the loaded mass was reduced (for all future SPE analyses as well) we no longer observed any loss of glucuronide during loading. A water wash was also inserted in between the 2% (v/v) aqueous formic acid and methanol fractions when glucuronide was observed to elute in the methanol fraction, with the concern that some of the glucuronide was still protonated and could therefore co-elute with the parent steroid 19. A subsequent purification using this protocol and cartridge afforded clean isolation of the glucuronide with no observed parent steroid contaminant.

Figure 9: The four protocols examined for solid phase extraction of glucuronides

The ARFL protocol did not appear to match the degree of selective elution that was afforded by the WAX protocol, with elution of the glucuronide observed in both flow-through and methanol fractions (which also contained the parent steroid) as well as the final MeOH : EtOAc : formic acid fraction, and was not investigated further.

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The Bond Elut Certify II cartridges were then evaluated to see if they could also perform as well as the Oasis WAX cartridge in cleanly isolating the glucuronide product. The Bond Elut Certify II cartridge is also a mixed-mode cartridge with hydrophobic (C8) and strong anion exchange (SAX) functionalities present on the sorbent.

Using the Oasis MAX protocol it was observed that the parent steroid selectively eluted in the methanol fraction but the glucuronide eluted in all but the flow-through fractions, and so this protocol was not investigated further.

The ARFL protocol did not provide satisfactory results either, with the glucuronide observed to elute in the water fraction as well as the methanol fraction, which resulted in the contamination of the glucuronide with parent steroid. Therefore, this protocol was not investigated further. Finally, the use of the manufacturer’s protocol for the Certify II cartridge was examined. It was observed that the glucuronide eluted in the same fraction as the parent steroid and therefore it again could not be applied.

In summary, the Bond Elut Certify II cartridge was unable to isolate the glucuronide product cleanly, in contrast to the successful isolation of glucuronide product by the Oasis WAX cartridge.

A final test of the utility of the Oasis WAX cartridge was its effectiveness in purifying DHEA 3- glucuronide 21 (from a synthesis with DHEA 19) from a glucuronylsynthase reaction, with the reaction mixture loaded directly onto the cartridge. Using the modified Oasis WAX protocol (with the extra water wash) it was again observed that clean separation of glucuronide from parent steroid DHEA 19 was achieved, confirming its viability as the purification protocol for the glucuronylsynthase reaction. Consequently the Oasis WAX cartridge with modified protocol was used for purifying the glucuronylsynthase reaction.